Bicyclic inhibitors of histone deacetylase

ABSTRACT

Provided herein are compounds and pharmaceutically acceptable salts thereof, and pharmaceutical compositions thereof, which are useful in the treatment of conditions associated with inhibition of HDAC (e.g., HDAC2).

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/445,022 filed Jan. 11, 2017 and U.S. Provisional Application No.62/555,298 filed Sep. 7, 2017, the contents of each of which areincorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with government support under Small BusinessInnovation Research (SBIR) grant 1R43AG048651-01A1 awarded by theNational Institute of Health (NIH). The government has certain rights inthe invention.

BACKGROUND

Inhibitors of histone deacetylases (HDAC) have been shown to modulatetranscription and to induce cell growth arrest, differentiation andapoptosis. HDAC inhibitors also enhance the cytotoxic effects oftherapeutic agents used in cancer treatment, including radiation andchemotherapeutic drugs. Marks, P., Rifkind, R. A., Richon, V. M.,Breslow, R., Miller, T., Kelly, W. K. Histone deacetylases and cancer:causes and therapies. Nat Rev Cancer, 1, 194-202, (2001); and Marks, P.A., Richon, V. M., Miller, T., Kelly, W. K. Histone deacetylaseinhibitors. Adv Cancer Res, 91, 137-168, (2004). Moreover, recentevidence indicates that transcriptional dysregulation may contribute tothe molecular pathogenesis of certain neurodegenerative disorders, suchas Huntington's disease, spinal muscular atrophy, amyotropic lateralsclerosis, and ischemia. Langley, B., Gensert, J. M., Beal, M. F.,Ratan, R. R. Remodeling chromatin and stress resistance in the centralnervous system: histone deacetylase inhibitors as novel and broadlyeffective neuroprotective agents. Curr Drug Targets CNS Neurol Disord,4, 41-50, (2005). A recent review has summarized the evidence thataberrant histone acetyltransferase (HAT) and histone deacetylases (HDAC)activity may represent a common underlying mechanism contributing toneurodegeneration. Moreover, using a mouse model of depression, Nestlerhas recently highlighted the therapeutic potential of histonedeacetylation inhibitors (HDACS) in depression. Tsankova, N. M., Berton,O., Renthal, W., Kumar, A., Neve, R. L., Nestler, E. J. Sustainedhippocampal chromatin regulation in a mouse model of depression andantidepressant action. Nat Neurosci, 9, 519-525, (2006).

There are 18 known human histone deacetylases, grouped into four classesbased on the structure of their accessory domains. Class I includesHDAC1, HDAC2, HDAC3, and HDAC8 and has homology to yeast Rpd3. HDAC4,HDACS, HDAC7, and HDAC9 belong to class IIa and have homology to yeastHda1. HDAC6 and HDAC10 contain two catalytic sites and are classified asclass IIb. Class III (the sirtuins) includes SIRT1, SIRT2, SIRT3, SIRT4,SIRT5, SIRT6, and SIRT7. HDAC11 is another recently identified member ofthe HDAC family and has conserved residues in its catalytic center thatare shared by both class I and class II deacetylases and is sometimesplaced in class IV.

HDACs have been shown to be powerful negative regulators of long-termmemory processes. Nonspecific HDAC inhibitors enhance synapticplasticity as well as long-term memory (Levenson et al., 2004, J. Biol.Chem. 279:40545-40559; Lattal et al., 2007, Behav Neurosci121:1125-1131; Vecsey et al., 2007, J. Neurosci 27:6128; Bredy, 2008,Learn Mem 15:460-467; Guan et al., 2009, Nature 459:55-60; Malvaez etal., 2010, Biol. Psychiatry 67:36-43; Roozendaal et al., 2010, J.Neurosci. 30:5037-5046). For example, HDAC inhibition can transform alearning event that does not lead to long-term memory into a learningevent that does result in significant long-term memory (Stefanko et al.,2009, Proc. Natl. Acad. Sci. USA 106:9447-9452). Furthermore, HDACinhibition can also generate a form of long-term memory that persistsbeyond the point at which normal memory fails. HDAC inhibitors have beenshown to ameliorate cognitive deficits in genetic models of Alzheimer'sdisease (Fischer et al., 2007, Nature 447:178-182; Kilgore et al., 2010,Neuropsychopharmacology 35:870-880). These demonstrations suggest thatmodulating memory via HDAC inhibition have considerable therapeuticpotential for many memory and cognitive disorders.

The role of individual HDACs in long-term memory has been explored intwo recent studies. Kilgore et al. 2010, Neuropsychopharmacology35:870-880 revealed that nonspecific HDAC inhibitors, such as sodiumbutyrate, inhibit class I HDACs (HDAC1, HDAC2, HDAC3, HDAC8) with littleeffect on the class IIa HDAC family members (HDAC4, HDACS, HDAC7,HDAC9). This suggests that inhibition of class I HDACs may be criticalfor the enhancement of cognition observed in many studies. Indeed,forebrain and neuron specific overexpression of HDAC2, but not HDAC1,decreased dendritic spine density, synaptic density, synaptic plasticityand memory formation. (Guan et al., 2009, Nature, 459:55-60). Incontrast, HDAC2 knockout mice exhibited increased synaptic density,increased synaptic plasticity and increased dendritic density inneurons. These HDAC2 deficient mice also exhibited enhanced learning andmemory in a battery of learning behavioral paradigms. This workdemonstrates that HDAC2 is a key regulator of synaptogenesis andsynaptic plasticity. Additionally, Guan et al. showed that chronictreatment of mice with SAHA (an HDAC 1, 2, 3, 6, 8 inhibitor) reproducedthe effects seen in the HDAC2 deficient mice and rescued the cognitiveimpairment in the HDAC2 overexpressing mice.

The inhibition of HDAC2 (selectively or in combination with inhibitionof other class I HDACs; as the primary target, or as part of a complexwith other proteins) is an attractive therapeutic target. Selectiveinhibition might be achieved by targeting specific HDAC isoforms such asHDAC2, in isolation, or as part of a functional multi-protein complex.Such inhibition has the potential for enhancing cognition andfacilitating the learning process through increasing synaptic anddendritic density in neuronal cell populations. In addition, inhibitionof specific HDACs, such as HDAC2, may also be therapeutically useful intreating a wide variety of other diseases and disorders.

SUMMARY

Disclosed are compounds and pharmaceutically acceptable salts thereof,and pharmaceutical compositions, which are useful in the treatment ofconditions associated with the activity of HDAC (e.g., HDAC2). (Seee.g., Tables 1 and 2).

The disclosed compounds provide an advantage in hematological safety andoverall balance of potency, ADME and PK profiles when compared to priorinhibitors. For example, the mere replacement of hydrogen for methylbetween Comparator E and Compound 2 leads to a dramatic decrease inCYP2D6 inhibition. See e.g., Table 2. Also, this replacement providesdistinct PK benefits over the unsubstituted pyrimidine analog,displaying a longer half-life, lower clearance, higher bioavailability,and a >5-fold higher brain exposure. See e.g., Table 3. Similarly, theaddition of one additional ortho-fluorine atom realized a significantsafety benefit in both the erythroid and myeloid progenitor celllineages for Compound 1 relative to Comparator I. See e.g., Table 5.

The described compounds also produce changes in dendritic spinemorphology in the CA1 region of the dorsal hippocampus in wild typemice. See e.g., Table 7. Measures of dendritic spine morphology canidentify pharmacological agents which are likely to promote or distortnormal cognitive function and protect against or exacerbate cognitiveimpairments.

Conditions which are treatable by the disclosed compounds include, butare not limited to, neurological disorders, memory or cognitive functiondisorders or impairments, extinction learning disorders, fungal diseasesor infections, inflammatory diseases, hematological diseases, neoplasticdiseases, psychiatric disorders, and memory loss.

DETAILED DESCRIPTION 1. Compounds

Provided herein is a compound of the formula:

or a pharmaceutically acceptable salt thereof.

2. Definitions

As used herein the terms “subject” and “patient” may be usedinterchangeably, and means a mammal in need of treatment, e.g.,companion animals (e.g., dogs, cats, and the like), farm animals (e.g.,cows, pigs, horses, sheep, goats and the like) and laboratory animals(e.g., rats, mice, guinea pigs and the like). Typically, the subject isa human in need of treatment.

Pharmaceutically acceptable salts as well as the neutral forms of thecompounds described herein are included. For use in medicines, the saltsof the compounds refer to non-toxic “pharmaceutically acceptable salts.”Pharmaceutically acceptable salt forms include pharmaceuticallyacceptable acidic/anionic or basic/cationic salts. Pharmaceuticallyacceptable basic/cationic salts include, the sodium, potassium, calcium,magnesium, diethanolamine, n-methyl-D-glucamine, L-lysine, L-arginine,ammonium, ethanolamine, piperazine and triethanolamine salts.Pharmaceutically acceptable acidic/anionic salts include, e.g., theacetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, carbonate,citrate, dihydrochloride, gluconate, glutamate, glycollylarsanilate,hexylresorcinate, hydrobromide, hydrochloride, malate, maleate,malonate, mesylate, nitrate, salicylate, stearate, succinate, sulfate,tartrate, and tosylate.

The term “pharmaceutically acceptable carrier” refers to a non-toxiccarrier, adjuvant, or vehicle that does not destroy the pharmacologicalactivity of the compound with which it is formulated. Pharmaceuticallyacceptable carriers, adjuvants or vehicles that may be used in thecompositions described herein include, but are not limited to, ionexchangers, alumina, aluminum stearate, lecithin, serum proteins, suchas human serum albumin, buffer substances such as phosphates, glycine,sorbic acid, potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts or electrolytes, such as protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol,sodium carboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat.

The terms “treatment,” “treat,” and “treating” refer to reversing,alleviating, reducing the likelihood of developing, or inhibiting theprogress of a disease or disorder, or one or more symptoms thereof, asdescribed herein. In some embodiments, treatment may be administeredafter one or more symptoms have developed, i.e., therapeutic treatment.In other embodiments, treatment may be administered in the absence ofsymptoms. For example, treatment may be administered to a susceptibleindividual prior to the onset of symptoms (e.g., in light of a historyof symptoms and/or in light of genetic or other susceptibility factors),i.e., prophylactic treatment. Treatment may also be continued aftersymptoms have resolved, for example to prevent or delay theirrecurrence.

The term “effective amount” or “therapeutically effective amount”includes an amount of a compound described herein that will elicit abiological or medical response of a subject.

3. Uses, Formulation and Administration

In some embodiments, compounds and compositions described herein areuseful in treating conditions associated with the activity of HDAC. Suchconditions include for example, those described below.

Recent reports have detailed the importance of histone acetylation incentral nervous system (“CNS”) functions such as neuronaldifferentiation, memory formation, drug addiction, and depression(Citrome, Psychopharmacol. Bull. 2003, 37, Suppl. 2, 74-88; Johannessen,CNS Drug Rev. 2003, 9, 199-216; Tsankova et al., 2006, Nat. Neurosci. 9,519-525; Bousiges et al., 2013, PLoS ONE 8(3), e57816). Thus, in oneaspect, the provided compounds and compositions may be useful intreating a neurological disorder. Examples of neurological disordersinclude: (i) chronic neurodegenerative diseases such as fronto-temporallobar degeneration (frontotemporal dementia, FTD), FTD-GRN, familial andsporadic amyotrophic lateral sclerosis (FALS and ALS, respectively),familial and sporadic Parkinson's disease, Parkinson's disease dementia,Huntington's disease, familial and sporadic Alzheimer's disease,multiple sclerosis, muscular dystrophy, olivopontocerebellar atrophy,multiple system atrophy, Wilson's disease, progressive supranuclearpalsy, diffuse Lewy body disease, corticodentatonigral degeneration,progressive familial myoclonic epilepsy, striatonigral degeneration,torsion dystonia, familial tremor, Down's Syndrome, Gilles de laTourette syndrome, Hallervorden-Spatz disease, diabetic peripheralneuropathy, dementia pugilistica, AIDS Dementia, age related dementia,age associated memory impairment, and amyloidosis-relatedneurodegenerative diseases such as those caused by the prion protein(PrP) which is associated with transmissible spongiform encephalopathy(Creutzfeldt-Jakob disease, Gerstmann-Straussler-Scheinker syndrome,scrapie, and kuru), and those caused by excess cystatin C accumulation(hereditary cystatin C angiopathy); and (ii) acute neurodegenerativedisorders such as traumatic brain injury (e.g., surgery-related braininjury), cerebral edema, peripheral nerve damage, spinal cord injury,Leigh's disease, Guillain-Barre syndrome, lysosomal storage disorderssuch as lipofuscinosis, Alper's disease, restless leg syndrome, vertigoas result of CNS degeneration; pathologies arising with chronic alcoholor drug abuse including, for example, the degeneration of neurons inlocus coeruleus and cerebellum, drug-induced movement disorders;pathologies arising with aging including degeneration of cerebellarneurons and cortical neurons leading to cognitive and motor impairments;and pathologies arising with chronic amphetamine abuse to includingdegeneration of basal ganglia neurons leading to motor impairments;pathological changes resulting from focal trauma such as stroke, focalischemia, vascular insufficiency, hypoxic-ischemic encephalopathy,hyperglycemia, hypoglycemia or direct trauma; pathologies arising as anegative side-effect of therapeutic drugs and treatments (e.g.,degeneration of cingulate and entorhinal cortex neurons in response toanticonvulsant doses of antagonists of the NMDA class of glutamatereceptor) and Wernicke-Korsakoff's related dementia. Neurologicaldisorders affecting sensory neurons include Friedreich's ataxia,diabetes, peripheral neuropathy, and retinal neuronal degeneration.Other neurological disorders include nerve injury or trauma associatedwith spinal cord injury. Neurological disorders of limbic and corticalsystems include cerebral amyloidosis, Pick's atrophy, and Rett syndrome.In another aspect, neurological disorders include disorders of mood,such as affective disorders and anxiety; disorders of social behavior,such as character defects and personality disorders; disorders oflearning, memory, and intelligence, such as mental retardation anddementia. Thus, in one aspect the disclosed compounds and compositionsmay be useful in treating schizophrenia, delirium, attention deficithyperactivity disorder (ADHD), schizoaffective disorder, Alzheimer'sdisease, vascular dementia, Rubinstein-Taybi syndrome, depression,mania, attention deficit disorders, drug addiction, dementia, dementiaincluding BPSD manifestations, agitation, apathy, anxiety, psychoses,personality disorders, bipolar disorders, unipolar affective disorder,obsessive-compulsive disorders, eating disorders, post-traumatic stressdisorders, irritability, adolescent conduct disorder and disinhibition.They may also be useful for spontaneous, toxic, neoplastic,post-traumatic and post-infectious tinnitus and smelling impairment.

Transcription is thought to be a key step for long-term memory formation(Alberini, 2009, Physiol. Rev. 89, 121-145). Transcription is promotedby specific chromatin modifications, such as histone acetylation, whichmodulate histone—DNA interactions (Kouzarides, 2007, Cell, 128:693-705),as well as transcription factor-DNA interactions. Modifying enzymes,such as histone acetyltransferases (HATs) and histone deacetylases(HDACs), regulate the state of acetylation on histone tails. In general,histone acetylation promotes gene expression, whereas histonedeacetylation leads to gene silencing, although treatment with HDACinhibitors can result in both upregulation and downregulation of theexpression levels of specific genes. Numerous studies have shown that apotent HAT, cAMP response element-binding protein (CREB)-binding protein(CBP), is necessary for long-lasting forms of synaptic plasticity andlong term memory (for review, see Barrett, 2008, Learn Mem 15:460-467).Thus, in one aspect, the provided compounds and compositions may beuseful for promoting cognitive function and enhancing learning andmemory formation.

The compounds and compositions described herein may also be used fortreating fungal diseases or infections.

In another aspect, the compounds and compositions described herein maybe used for treating inflammatory diseases such as stroke, rheumatoidarthritis, lupus erythematosus, ulcerative colitis and traumatic braininjuries (Leoni et al., PNAS, 99(5); 2995-3000(2002); Suuronen et al. J.Neurochem. 87; 407-416 (2003) and Drug Discovery Today, 10: 197-204(2005).

In yet another aspect, the compounds and compositions described hereinmay be used for treating a cancer caused by the proliferation ofneoplastic cells. Such cancers include e.g., solid tumors, neoplasms,carcinomas, sarcomas, leukemias, lymphomas and the like. In one aspect,cancers that may be treated by the compounds and compositions describedherein include, but are not limited to: cardiac cancer, lung cancer,gastrointestinal cancer, genitourinary tract cancer, liver cancer,nervous system cancer, gynecological cancer, hematologic cancer, skincancer, and adrenal gland cancer. In one aspect, the compounds andcompositions described herein are useful in treating cardiac cancersselected from sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma,liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma and teratoma. Inanother aspect, the compounds and compositions described herein areuseful in treating a lung cancer selected from bronchogenic carcinoma(squamous cell, undifferentiated small cell, undifferentiated largecell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchialadenoma, sarcoma, lymphoma, chondromatous hamartoma, and mesothelioma.In one aspect, the compounds and compositions described herein areuseful in treating a gastrointestinal cancer selected from esophagus(squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma),stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (ductaladenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors,vipoma), small bowel (adenocarcinoma, lymphoma, carcinoid tumors,Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma),and large bowel (adenocarcinoma, tubular adenoma, villous adenoma,hamartoma, leiomyoma). In one aspect, the compounds and compositionsdescribed herein are useful in treating a genitourinary tract cancerselected from kidney (adenocarcinoma, Wilm's tumor [nephroblastoma],lymphoma, leukemia), bladder and urethra (squamous cell carcinoma,transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma,sarcoma), and testis (seminoma, teratoma, embryonal carcinoma,teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma,fibroma, fibroadenoma, adenomatoid tumors, lipoma). In one aspect, thecompounds and compositions described herein are useful in treating aliver cancer selected from hepatoma (hepatocellular carcinoma),cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellularadenoma, and hemangioma.

In some embodiments, the compounds described herein relate to treating,a bone cancer selected from osteogenic sarcoma (osteosarcoma),fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing'ssarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma,malignant giant cell tumor chordoma, osteochondroma (osteocartilaginousexostoses), benign chondroma, chondroblastoma, chondromyxofibroma,osteoid osteoma and giant cell tumors.

In one aspect, the compounds and compositions described herein areuseful in treating a nervous system cancer selected from skull (osteoma,hemangioma, granuloma, xanthoma, osteitis deformans), meninges(meningioma, meningiosarcoma, gliomatosis), brain (astrocytoma,medulloblastoma, glioma, ependymoma, germinoma [pinealoma], glioblastomamultiform, oligodendroglioma, schwannoma, retinoblastoma, congenitaltumors), and spinal cord (neurofibroma, meningioma, glioma, sarcoma).

In one aspect, the compounds and compositions described herein areuseful in treating a gynecological cancer selected from uterus(endometrial carcinoma), cervix (cervical carcinoma, pre-tumor cervicaldysplasia), ovaries (ovarian carcinoma [serous cystadenocarcinoma,mucinous cystadenocarcinoma, unclassified carcinoma], granulosa-thecalcell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignantteratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma,adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma,squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma),and fallopian tubes (carcinoma).

In one aspect, the compounds and compositions described herein areuseful in treating a skin cancer selected from malignant melanoma, basalcell carcinoma, squamous cell carcinoma, Karposi's sarcoma, molesdysplastic nevi, lipoma, angioma, dermatofibroma, keloids, andpsoriasis.

In one aspect, the compounds and compositions described herein areuseful in treating an adrenal gland cancer selected from neuroblastoma.

In one aspect, the compounds and compositions described herein areuseful in treating cancers that include, but are not limited to:leukemias including acute leukemias and chronic leukemias such as acutelymphocytic leukemia (ALL), Acute myeloid leukemia (AML), chroniclymphocytic leukemia (CLL), chronic myelogenous leukemia (CML) and HairyCell Leukemia; lymphomas such as cutaneous T-cell lymphomas (CTCL),noncutaneous peripheral T-cell lymphomas, lymphomas associated withhuman T-cell lymphotrophic virus (HTLV) such as adult T-cellleukemia/lymphoma (ATLL), Hodgkin's disease and non-Hodgkin's lymphomas,large-cell lymphomas, diffuse large B-cell lymphoma (DLBCL); Burkitt'slymphoma; mesothelioma, primary central nervous system (CNS) lymphoma;multiple myeloma; childhood solid tumors such as brain tumors,neuroblastoma, retinoblastoma, Wilm's tumor, bone tumors, andsoft-tissue sarcomas, common solid tumors of adults such as head andneck cancers (e.g., oral, laryngeal and esophageal), genito urinarycancers (e.g., prostate, bladder, renal, uterine, ovarian, testicular,rectal and colon), lung cancer, breast cancer, pancreatic cancer,melanoma and other skin cancers, stomach cancer, brain tumors, livercancer and thyroid cancer.

In one aspect, the compounds and compositions described herein areuseful in treating a condition in a subject selected from a neurologicaldisorder, memory or cognitive function disorder or impairment,extinction learning disorder, fungal disease or infection, inflammatorydisease, hematological disease, psychiatric disorders, and neoplasticdisease. In another aspect, the compounds and compositions describedherein are useful in treating a condition selected from a) a cognitivefunction disorder or impairment associated with Alzheimer's disease,Huntington's disease, seizure induced memory loss, schizophrenia,Rubinstein Taybi syndrome, Rett Syndrome, Fragile X, Lewy body dementia,vascular dementia, fronto-temporal lobar degeneration (frontotemporaldementia, FTD), FTD-GRN, ADHD, dyslexia, bipolar disorder and social,cognitive and learning disorders associated with autism, traumatic headinjury, attention deficit disorder, anxiety disorder, conditioned fearresponse, panic disorder, obsessive compulsive disorder, posttraumaticstress disorder (PTSD), phobia, social anxiety disorder, substancedependence recovery, Age Associated Memory Impairment (AAMI), AgeRelated Cognitive Decline (ARCD), ataxia, or Parkinson's disease; b) ahematological disease selected from acute myeloid leukemia, acutepromyelocytic leukemia, acute lymphoblastic leukemia, chronicmyelogenous leukemia, myelodysplastic syndromes, and sickle cell anemia;c) a neoplastic disease; and d) an extinction learning disorder selectedfrom fear extinction and post-traumatic stress disorder. In one aspect,the condition treated by the compounds and compositions described hereinis Alzheimer's disease, Huntington's disease, frontotemporal dementia,Freidreich's ataxia, post-traumatic stress disorder (PTSD), Parkinson'sdisease, depression, or substance dependence recovery.

In one aspect, the present disclosure provides a method of treating acondition described herein comprising administering to a subject aneffective amount of a compound, or pharmaceutically acceptable saltdescribed herein, or a composition thereof.

Also provided is the use of one or more of the compounds, orpharmaceutically acceptable salts thereof described herein, or aprovided composition, for treating a condition described herein.

Also provided is the use of one or more of the compounds, orpharmaceutically acceptable salts thereof described herein for themanufacture of a medicament for treating a condition described herein.

Subjects may also be selected to be suffering from one or more of thedescribed conditions before treatment with one or more of the describedcompounds, or pharmaceutically acceptable salts or compositionscommences.

The present disclosure also provides pharmaceutically acceptablecompositions comprising a compound described herein, or apharmaceutically acceptable salt thereof; and a pharmaceuticallyacceptable carrier. These compositions can be used to treat one or moreof the conditions described above.

Compositions described herein may be administered orally, parenterally,by inhalation spray, topically, rectally, nasally, buccally, vaginallyor via an implanted reservoir. The term “parenteral” as used hereinincludes subcutaneous, intravenous, intramuscular, intra-articular,intra-synovial, intrasternal, intrathecal, intrahepatic, intralesionaland intracranial injection or infusion techniques. Liquid dosage forms,injectable preparations, solid dispersion forms, and dosage forms fortopical or transdermal administration of a compound are included herein.

The amount of provided compounds that may be combined with carriermaterials to produce a composition in a single dosage form will varydepending upon the patient to be treated and the particular mode ofadministration. In some embodiments, provided compositions may beformulated so that a dosage of between 0.01-100 mg/kg body weight/day ofthe provided compound, such as e.g., 0.1-100 mg/kg body weight/day, canbe administered to a patient receiving these compositions.

It should also be understood that a specific dosage and treatmentregimen for any particular patient will depend upon a variety offactors, including age, body weight, general health, sex, diet, time ofadministration, rate of excretion, drug combination, the judgment of thetreating physician, and the severity of the particular disease beingtreated. The amount of a provided compound in the composition will alsodepend upon the particular compound in the composition.

EXEMPLIFICATION

Spots were visualized by UV light (254 and 365 nm). Purification bycolumn and flash chromatography was carried out using silica gel(200-300 mesh). Solvent systems are reported as the ratio of solvents.

NMR spectra were recorded on a Bruker 400 (400 MHz) spectrometer. ¹Hchemical shifts are reported in δ values in ppm with tetramethylsilane(TMS, =0.00 ppm) as the internal standard. See, e.g., the data providedin Table 1.

LCMS spectra were obtained on an Agilent 1200 series 6110 or 6120 massspectrometer with ESI (+) ionization mode. See, e.g., the data providedin Table 1.

Example 1

Synthesis of SM-A.

A mixture of 6-chloro-3-nitropyridin-2-amine (4.58 g, 26.4 mmol),2,4-difluorophenylboronic acid (5.00 g, 31.7 mmol) and Cs₂CO₃ (25.73 g,79.2 mmol) in dioxane/H₂O (100 mL/10 mL) was added Pd(PPh₃)₄ (1.10 g,0.95 mmol) under N₂ atmosphere. The mixture was stirred at 100° C. for 2h and then concentrated in vacuo. The residue was dissolved with EtOAc(200 mL) and the solution was washed with brine (100 mL×3). The organiclayer was dried over anhydrous Na₂SO₄ and then concentrated in vacuo.The residue was purified by column chromatography on silica gel(PE:EtOAc=7:1˜5:1) to give SM-A (4.0 g, 61%) as a yellow solid. MS 252.1[M+H]⁺.

Synthesis of SM-B.

To a stirring solution of SM-A (4.0 g, 15.94 mmol) in pyridine (60 mL)was added phenyl carbonochloridate (7.50 g, 47.81 mmol) dropwise at 0°C. After the addition was completed, the mixture was stirred at 50° C.for 4 h. The mixture was concentrated in vacuo. The residue was purifiedby column chromatography on silica gel (PE:DCM=3:2˜1:1) to give SM-B(7.1 g, 91%) as a yellow solid. MS 492.1 [M+H]⁺.

Synthesis of 1672-1.

To a solution of prop-2-yn-1-amine (5.0 g, 90.9 mmol) and Et₃N (18.4 g,181.8 mmol) in DCM (100 mL) was added (Boc)₂O (23.8 g, 109.1 mmol)dropwise while cooling the reaction mixture with an ice bath. Theresulting mixture was removed from the ice bath once the addition wascompleted, and was then stirred at room temperature for 16 h. When thereaction was complete, the mixture was diluted with DCM (200 mL), washedwith brine (100 mL×3), and the organic layer was then dried over Na₂SO₄and then concentrated in vacuo. The residue was purified by columnchromatography on silica gel (PE:EtOAc=100:1˜10:1) to give 1672-1 (10 g,71%) as a colorless oil. MS 178.3 [M+23]⁺, 100.3 [M−56]⁺.

Synthesis of 1672-2.

To a solution of 1672-1 (10 g, 64.5 mmol) in DMF (200 mL) was added NaH(60% in mineral oil) (2.84 g, 71 mmol) slowly under ice bath. Theresulting mixture was stirred at room temperature for 1 h, whereupon3-bromoprop-1-yne (9.2 g, 77.4 mmol) was added into above mixture, andthe reaction mixture was then stirred at room temperature for 2 h. Themixture was quenched with water (500 mL) and then extracted with t-BuOMe(250 mL×3). The combined organic layers were washed with brine (200mL×3), dried over anhydrous Na₂SO₄ and then concentrated in vacuo. Theresidue was purified by column chromatography on silica gel(PE:EtOAc=100:1˜10:1) to give 1672-2 (12 g, 96%) as a yellow oil. MS138.1 [M−56]⁺.

Synthesis of 1672-3.

To a solution of 2-chloroacetonitrile (3.13 g, 41.4 mmol) and[Cp*RuCl(cod)] (394 mg, 1.0 mmol) in DCE (40 mL) was added a solution of1672-2 (4.0 g, 20.7 mmol) in DCE (80 mL) dropwise over 30 min under anN₂ atmosphere. The resulting mixture was stirred at 40 ° C. for 16 h.The solvent was removed in vacuo, and the residue was purified by columnchromatography on silica gel (PE:EtOAc=10:1˜2:1) to give 1672-3 (2.1 g,22%) as a khaki solid. MS 269.3 [M+H]⁺.

Synthesis of 1672-4.

To a solution of MeOH (30 mL) was added NaH (60% in mineral oil) (940mg, 23.5 mmol) at ice bath and stirred for 30 min. Then 1672-C (2.1 g,7.8 mmol) was added into above mixture and stirred at 35° C. for 16 h.The mixture was quenched with water (30 mL), extracted with DCM (10mL×3). The combined organic layers were washed with brine (10 mL×3),dried over anhydrous Na₂SO₄ and then concentrated in vacuo. The residuewas purified by column chromatography on silica gel(PE:EtOAc=100:1˜10:1) to give 1672-4 (1.8 g, 94%) as a tan coloredsolid. MS 265.1 [M+H]⁺.

Synthesis of 1672-5.

To a solution of 1672-4 (120 mg, 0.45 mmol) in DCM (6 mL) cooled in anice bath was added TFA (2 mL) dropwise. The resulting reaction mixturewas stirred at room temperature for 1 h, whereupon the solvent wasremoved in vacuo to give 1672-5 as a crude product which was taken on tothe next step without further purification. MS 165.1 [M+H]⁺.

Synthesis of 1672-6.

To a mixture of 1672-5 (0.45 mmol, crude product from last step) andSM-B (150 mg, 0.30 mmol) in DMSO (10 mL) was added Na₂CO₃ (259 mg, 3.44mmol), and the resulting reaction mixture was stirred at 25° C. for 2 h.The mixture was then diluted with water (30 mL) and extracted with EtOAc(20 mL×3). The combined organic layers were washed with brine (10 mL×3),dried over anhydrous Na₂SO₄ and then concentrated in vacuo. The residuewas purified by column chromatography on silica gel(DCM:MeOH=100:1˜30:1) to give 1672-6 (120 mg, 89%) as a yellow solid. MS442.1 [M+H]₊.

Synthesis of Compound 1.

A mixture of 1672-6 (120 mg, 0.27 mmol) and Pd/C (120 mg) in MeOH (10mL) was stirred at room temperature for 1 h under a H₂ atmosphere. Pd/Cwas then removed by filtration through the celite. The filtrate wasconcentrated and the residue was purified by Prep-TLC (DCM:MeOH=15:1) togive Compound 1 (70 mg, 70%) as a yellow solid. MS 412.1 [M+H]⁺, 434.1[M+23]⁺.

Example 2

Synthesis of 156-A.

A mixture of 6-chloro-3-nitropyridin-2-amine (10.00 g, 57.6 mmol),thiophen-2-ylboronic acid (8.12 g, 63.4 mmol) and Cs₂CO₃ (37.56 g, 115.2mmol) in dioxane/H₂O (200 mL/20 mL) was added Pd(PPh₃)₄ (2.44g, 2.88mmol) under an N₂ atmosphere. The mixture was stirred at 95° C. for 2 hand then concentrated in vacuo. The residue was dissolved with EtOAc(200 mL) and the solution was washed with brine (100 mL×3). The organiclayer was dried over anhydrous Na₂SO₄ and then concentrated in vacuo.The residue was purified by column chromatography on silica gel(PE:EtOAc=5:1˜3:1) to give 156-A (10.0 g, 79%) as a yellow solid

Synthesis of 156-B.

To a stirred solution of 156-A (1.30 g, 5.88 mmol) in pyridine (20 mL)was added phenyl carbonochloridate (2.29 g, 14.7 mmol) in dropwisefashion. After the addition was completed, the mixture was heated to 50°C. and stirred for 4 h. The mixture was then concentrated in vacuo, andthe residue was purified by column chromatography on silica gel(PE:EtOAc=8:1˜3:1) to give 156-B (2.4 g, 89%) as a yellow solid.

Synthesis of 213-A.

A solution of tert-butyl 3-oxopyrrolidine-1-carboxylate (150.0 g, 809.8mmol) and DMF-DMA (289.5 g, 2.4 mol) in THF (1500 mL) was stirred at 70°C. for 16 h. The solution was concentrated in vacuo to give 213-A as acrude product, which was used directly in the next step without furtherpurification.

Synthesis of 213-B.

To a solution of 213-A (809.8 mmol, crude product from last step) inEtOH (1000 mL) was added Et₃N (409.7 g, 4.0 mol) and acetimidamidehydrochloride (306.2 g, 3.2 mol). The resulting solution was stirred at80° C. for 24 h. After the mixture was cooled to room temperature, themixture was diluted with water (500 mL) and extracted with DCM (500mL×3). The combined organic layers were washed with brine (500 mL×3),dried over anhydrous Na₂SO₄ and then concentrated in vacuo. The residuewas purified by column chromatography on silica gel (PE:DCM=10:1˜1:2) togive 213-B (105.0 g, 55%) as a brown solid.

Synthesis of 213-C.

To a solution of 213-B (105.0 g, 446.3 mmol) in DCM (1000 mL) was addedTFA (333 mL) dropwise. The reaction mixture was stirred at roomtemperature for 1 h, whereupon the solution was concentrated in vacuo togive 213-C as a crude product which was used directly in the next step.

Synthesis of 213-D.

A mixture of 213-C (325.1 mmol, crude product from last step) and 156-B(75.0 g, 162.5 mmol) in DMSO (750 mL) was stirred at room temperaturefor 10 min, then Na₂CO₃ (137.8 g, 1.3 mol) was added, and the reactionmixture was stirred at room temperature for 2 h. The mixture was thendiluted with water (1000 mL) and extracted with EtOAc (500 mL×3). Thecombined organic layers were washed with brine (500 mL×3), dried overanhydrous Na₂SO₄ and then concentrated in vacuo. The residue waspurified by column chromatography on silica gel (PE:EA=10:1˜1:2) to give213-D (44.0 g, 71%) as a yellow solid.

Synthesis of Compound 2.

A mixture of 213-D (44.0 g, 115.1 mmol) and Pd/C (22.0 g) in MeOH (250mL) and DCM (250 mL) was stirred at room temperature for 1 h under a H₂atmosphere. Pd/C was removed by filtration through Celite. The filtratewas concentrated in vacuo and the residue was purified by columnchromatography on silica gel (DCM:MeOH=50:1˜15:1) to give Compound 2(26.0 g, 64%) as a light yellow solid.

TABLE 1 Spectrometric Data for Compounds MS MS ¹H NMR Data (400 MHz, No.Structure Calc found DMSO-d₆) 1

411 412 δ 8.58 (s, 1H), 8.53 (s, 1H), 7.97- 7.91 (m, 1H), 7.44-7.40 (m,2H), 7.32-7.26 (m, 1H), 7.18-7.13 (m, 2H), 5.28 (s, 2H), 4.82 (s, 4H),4.52 (s, 2H), 3.38 (s, 3H). 2

352 353 δ 8.70 (s, 1H), 8.60 (s, 1H), 7.53- 7.47 (m, 2H), 7.42-7.40 (q,J = 4.0 Hz, 1H), 7.13-7.07 (t, J = 8.0 Hz, 1H), 7.06-7.05 (t, J = 1.2Hz, 1H), 5.18 (s, 2H), 4.78-4.75 (d, J = 10.4 Hz, 4H), 2.64 (s, 3H).

General Assay Methods HDAC2 and HDAC1 Enzymatic Assay

The following describes an assay protocol for measuring thedeacetylation of a peptide substrate by the enzymes HDAC2 or HDAC1.

All recombinant human HDACs were purchased from BPS Bioscience. Thesubstrate, FAM-TSRHK(AC)KL-CONH, was synthesized at NanoSyn. Final assayreactions contained 100 mM HEPES (pH 7.5), 50 mM KCl, 0.1% BSA, 0.01%Triton X-100, 1% DMSO, 1 uM substrate and 5 nM HDAC enzyme. Enzyme andcompounds were pre-incubated at 25° C. for 5 hours and reactions wereinitiated by addition of substrate. 10 uL reactions were incubated for17 hours at 25° C. and terminated by the addition of 40 uL of buffercontaining 100 mM HEPES (pH 7.5), 0.1% BSA, 0.01% Triton X-100 and 0.05%SDS. Substrate and product peptides present in each sample wereseparated electrophoretically using the LabChip 3000 capillaryelectrophoresis instrument. Change in the relative fluorescenceintensity of the substrate and product peaks reflects enzyme activity.Reaction progress was determined as the product to sum ratio(PSR):P/(S+P), where P is the peak height of the product peptide and Sis the peak height of the substrate peptide. Reactions were performed induplicate at 12 concentrations, (3× serial dilutions starting at 30 uM).IC₅₀ values were calculated using a 4 Parameter Logistic Model.

HDAC2 Enzymatic Inhibition Assay in SY5Y Cell Lysate with HDAC-Glo2Substrate Cell Culture and Inhibitor Treatments

SH-SYSY cells (Sigma) were cultured in Eagle's Modified Essential Mediumsupplemented with 10% fetal bovine serum and pen/strep. Twenty-fourhours prior to compound dosing 20 uL of cells were plated in white 384well plates at a density of 1,500 cells/well. Compounds were seriallydiluted in neat DMSO and then diluted 1:100 v/v into media without FBSand mixed. Media was removed from the plated cells and the dilutedcompounds in serum free media (1% v/v final DMSO) were added andincubated at 37.0 for five hours. Ten uL of HDAC-Glo 2 reagent with 0.1%Triton X-100 was then added, the plate was mixed and allowed to developat room temperature for 100 minutes. Plates were then read with aSpectramax LMax luminometer employing a 0.4s integration time. Doseresponse curves were constructed with normalized data where CI-994 at100 uM was defined as 100% inhibition and DMSO alone as 0% inhibition.

Erythroid and Myeloid CFU Assay

Compounds were tested to evaluate the potential effects on humanerythroid and myeloid progenitors using colony forming cell assays.Clonogenic progenitors of human erythroid (CFU-E, BFU-E),granulocyte-monocyte (CFU-GM) and multipotential (CFU-GEMM) lineageswere assessed in a semi-solid methylcellulose-based media formulationcontaining rhIL-3 (10 ng/mL), rhGM-SCF (10 ng/mL), rhSCF (50 ng/mL) andEpo (3 U/mL).

Cells

Normal human bone marrow light density cells derived from normal bonemarrow (NorCal Biologics, California) and qualified at ReachBio, werestored in the gaseous phase of liquid nitrogen (−152° C.) until requiredfor the assay. On the day of the experiment, the cells were thawedrapidly, the contents of each vial was diluted in 10 mL of Iscove'smodified Dulbecco's medium containing 10% fetal bovine serum (IMDM+10%FBS) and washed by centrifugation (approximately 1200 r.p.m. for 10minutes, room temperature). The supernatant was discarded and the cellpellets resuspended in a known volume of IMDM+10% FBS. A cell count (3%glacial acetic acid) and viability assessment (trypan blue exclusiontest) was performed for the bone marrow sample.

Compounds

On the day of the experiment, the compounds were dissolved in DMSO to astock concentration of 10 mM. Serial dilutions were prepared from thestock concentration to achieve concentrations of 2 and 0.4 mM. Whenadded to the methylcellulose-based media at 1:1000 (v/v), the final testconcentrations of 10, 2 and 0.4 μM were achieved. Additionally, 5-FU wasevaluated at 1.0, 0.1 and 0.01 μg/mL.

Method Summary

Clonogenic progenitors of the human erythroid (CFU-E and BFU-E) andmyeloid (CFU-GM) lineages were set up in the methylcellulose-based mediaformulations described above. All compounds were added to the medium togive the final desired concentrations (10, 2 and 0.4 μM). 5-Fluorouracil(Sigma Aldrich) was used as a positive control for progenitorproliferation (inhibition of colony growth) and was introduced to thehuman bone marrow cultures at 1.0, 0.1, and 0.01 μg/mL. Solvent controlcultures (containing no compound but 0.1% DMSO) as well as standardcontrols (containing no compound and no DMSO) were also initiated.

Human myeloid and erythroid progenitor assays were initiated at 2.0×10⁴cells per culture. Following 14 days in culture, myeloid and erythroidcolonies were assessed microscopically and scored by trained personnel.The colonies were divided into the following categories based on sizeand morphology: CFU-E, BFU-E, CFU-GM and CFU-GEMM.

Statistical Analyses of CFC numbers

The mean±one standard deviation of three replicate cultures wascalculated for progenitors of each category (CFU-E, BFU-E, etc.).Two-tailed t-tests were performed to assess if there was a difference inthe number of colonies generated between solvent control and treatedcultures. Due to the potential subjectivity of colony enumeration, a pvalue of less than 0.01 is deemed significant. To calculate theconcentration of 50% inhibition of colony growth (IC₅₀) for eachcompound, a dose response curve was generated plotting the log of thecompound concentration versus the percentage of control colony growthusing XLfit software (IDBS). The concentration of 50% inhibition ofcolony growth (IC₅₀) was calculated based on the sigmoid curve fit usingDose-Response, One-Site Model formula: y=A+[(B−A)/(1+((C/x)̂D))], whereA=the initial value (baseline response), B=maximum response, C=center(drug concentration that provokes a response halfway between A and B)and D=slope of the curve at midpoint. Further, plots and additional doseresponse curves were generated using GraphPad Prism 7.0.

Morphological Assessment of Colonies

Photographs were taken of representative hematopoieticprogenitor-derived colonies from various lineages, illustrating coloniesin the presence of the solvent control as well as colonies in thepresence of the test compounds.

Erythroid (CFU-E and BFU-E), myeloid (CFU-GM) and multi-potential(CFU-GEMM) colony enumeration was performed by trained personnel. Thedistribution of colony types as well as general colony and cellularmorphology was analyzed. For statistical analysis colony numbers incompound treated cultures were compared to the solvent control cultures.5-FU was used as a positive control for toxicity in these assays and theinhibitory effects obtained for this compound were exactly as expected.The experiment was used to evaluate the potential effect of testcompounds on human erythroid and myeloid progenitor proliferation in amethylcellulose-based medium. The IC₅₀ values were calculated fromXLfit. Dose response curves for erythroid and myeloid toxicity generatedby XLfit. Finally, nonlinear regression curve fitting and IC₅₀s±95% CI,were calculated by Prism 7.0.-GEMM.

CYP Inhibition Assay

Compounds were tested to evaluate their inhibitory potential on CYP2D6and CYP3A4 (midazolam) using human liver microsomes. Human livermicrosomes were obtained from BD Gentest, and each compound was run induplicate.

The test compounds and reference inhibitors (quinidine for 2D6,ketoconazole for 3A4) were plated in a 96-well plate by transferring 8μL of 10 mM stock solutions of compound in DMSO to 12 μL ofacetonitrile. Individual inhibitor spiking solutions were prepared forCYP2D6 and CYP3A4 (8 μL of DMSO stock added to 12 μL of acetonitrile).Next added 400 μL of 0.2 mg/mL HLM to the assay wells and then added 2μL of 400×test compound into the designated wells on ice. Next, added200 μL of 0.2 mg/mL HLM to the assay wells and then added 1 μL ofreference inhibitor solutions into the designated wells. The followingsolutions were added (in duplicate) to a 96-well assay plate on ice: Thetest compounds and reference inhibitors (quinidine for 2D6, ketoconazolefor 3A4) were tested using the following experimental procedure:

1. Prepare test compound and reference inhibitors (400×) in a 96-wellplate:

1.1. Transfer 8 μL of 10 mM test compounds to 12 μL of ACN.

1.2. Prepare individual inhibitor spiking solution for CYP3A4, CYP2D6: 8μL of DMSO stock to 12 μL of ACN.

2. Prepare 4×NADPH cofactor (66.7 mg NADPH in 10 mL 0.1 M K-buffer, pH7.4)

3. Prepare 4×substrate (2 mL for each isoform) as indicated in the tablebelow (add HLM where required on ice).

4. Prepare 0.2 mg/mL HLM solution (10 μL of 20 mg/mL to 990 μL of 0.1 MK-buffer) on ice.

5. Add 400 μL of 0.2 mg/mL HLM to the assay wells and then add 2 μL of400×test compound into the designated wells on ice.

6. Add 200 μL of 0.2 mg/mL HLM to the assay wells and then add 1 μL ofreference inhibitor solution into the designated wells on ice.

7. Add following solutions (in duplicate) in a 96-well assay plate onice:

7.1. Add 30 μL of 2×test compound and reference compound in 0.2 mg/mLHLM solution;

7.2. Add 15 μL of 4×substrate solution.

8. Pre-incubate the 96-well assay plate and NADPH solution at 37° C. for5 minutes.

9. Add 15 μL of pre-warmed 8 mM NADPH solution to into the assay platesto initiate the reaction

10. Incubate the assay plate at 37° C. 5 min for 3A4, 10 min for 2D6.

11. Stop the reaction by adding 120 μL of ACN containing InternalStandard. For CYP3A4, the internal standard is 1′OH-midazolam-D₄ (10 μMsolution diluted to a final concentration of 0.1 μM by adding 100 μLinternal standard stock to 10 mL ACN). For CYP2D6, the internal standardis 1-OH-Bufuralol-maleate-[D₉] (49 μM solution diluted to a finalconcentration of 0.1 μM by adding 20 μL internal standard stock to 10 mLACN).

12. After quenching, shake the plates at the vibrator (IKA, MTS 2/4) for10 min (600 rpm/min) and then centrifuge at 5594 g for 15 min (ThermoMultifuge×3R).

13. Transfer 50 μL of the supernatant from each well into a 96-wellsample plate containing 50 μL of ultra pure water (Millipore, ZMQS50F01)for LC/MS analysis.

The assessment on CYP isoform inhibition is as follows based on theassay results: if percentage of CYP inhibition is higher than 50%,indicates potent inhibition; if percentage of CYP inhibition is between30-50%, indicates slight inhibition; if percentage of CYP inhibition isless than 30%, indicates slight or no inhibition. If the percentage ofCYP inhibition is less than −30%, this indicates the compound may havesome kind of activation of this isoform.

Aqueous Kinetic Solubility Measurement

Compounds were evaluated for their kinetic solubility in buffer orwater. Aliquots of 8 μL of reference and test compound stock solutions(10 mM in DMSO) were added into 792 μL of 100 mM phosphate buffer (0.1 MNaPO₄, pH 7.4). Final DMSO concentration is 1%. The sample tubes wereshaken for 1 hour (1000 rpm) at room temperature. A calibration curvewas prepared using 300 μM spiking solution (SS) in MeOH/ACN(4:1) (SS=add6 μL 10 mM compoud in 194 uL MeOH/ACN(4:1)). Samples were centrifugedfor 10 mM (12000 rpm) to precipitate undissolved particles, and thesupernatants were transferred to a new tube or plate. Supernatants werediluted 10 times and 100 times with 100 mM buffer. Samples were thenprepared for analysis by LC-MS/MS (Add 5 μL of compound samples (notdiluted, 10 times diluted and 100 times diluted) and standard curvesamples to 95 μL of ACN containing internal standard. Internal standardsused are Propranolol, Ketoconazole, and Tamoxifen.

Assessment of Brain and Plasma Exposure for Compounds FollowingIntravenous (IV) and Oral (PO) Administration to Mice

Compounds were dosed in mice at either 10 mg/kg or 30 mg/kg PO, and weredosed at 1 mg/kg IV. Three animals for collection at each time point forplasma via bleeding at 0.25, 0.5, 1, 4, 12 and 24 h. Terminal bleedingfor plasma and sampling for brain at 0.25, 0.5, 1, 4,12 and 24 h (alsothree animals per brain exposure time point group). Total of six timepoints for plasma and six time points for brain.

Sample Collection:

Plasma: The animal was restrained manually at the designated timepoints, approximately 150 μL blood/time point was collected into K₂EDTAtube via retro orbital puncture or cardiac puncture under anesthesiawith Isoflurane. The blood sample was centrifuged (2000 g, 4° C., 5 mM)to generate plasma within 30 mM after bleeding.

Brain: At the designated time points, a mid-line incision was made inthe animals scalp and the skin was retracted. Using small bone cuttersand rongeurs, removed the skull overlying the brain. Removed the brainusing a spatula and rinse with cold saline. Placed the brain inscrew-top tubes, and then stored the tubes under −70° C. until analysis.

Certain Advantages of Compounds 1 and 2

From a drug discovery standpoint, it is important that compounds haveacceptable drug-like profiles across a range of parameters. It istypical to profile compounds not only for in vitro potency, but also inpredictive Absorption, Distribution, Excretion and Metabolism (ADME)studies in vitro, and in pharmacokinetic (PK) experiments in vivo. Insome cases, compounds are also profiled in predictive in vitro safetystudies. Collecting in vitro ADME and safety data, along with PK data,help to identify benefits of certain structural features, and allows theoptimization of the structure activity relationship (SAR) to designcompounds with optimized drug-like profiles for profiling in vivo. Thepresent compounds not only provide an advantage in hematological safety,but also provide an overall balance of potency, ADME and PK profiles.

Aminoaniline urea compounds such as Comparator A and Comparator B inTable 2 were previously described in WO 2017/007755 and WO 2017/007756(the contents of each of which are incorporated herein by reference).When screened in an in vitro colony forming unit (CFU) assay in humanbone marrow cells, looking at erythroid and myeloid progenitor cells(predictive for neutropenia), and compared to the matched pairaminopyridine urea compound Comparator B, a single atom change in thediaminopyridine urea core of Comparator B leads to a significantimprovement in predicted safety in both erythroid and myeloid lineagesrelative to the urea of the aminoaniline scaffold of Comparator A. SeeTable 2. Moving forward, when the 4-fluorophenyl group of Comparator Bis replaced with a thiophene group (Comparator C), but thepyrrolopyrazine component of the urea is kept the same, a similarimprovement in predicted safety profile is achieved relative to theaminoaniline urea Comparator A. This evidences that ureas ofdiaminopyridine compounds with identical pyrrolidine urea components aresafer than the corresponding aminoaniline ureas.

While the pyrrolopyrazine urea compounds, Comparator B and Comparator C,showed good potency in in vitro potency assays, the values from the invitro CFU assay still require improvement. A myeloid lineage IC₅₀>5 μM(roughly corresponding to >30% remaining at 10 μM) predicts lowlikelihood of clinical neutropenia, so is the target threshold foracceptable safety (References: Pessina et al. Toxicological Sciences2003, 75, 355-367; Clarke et al. Gen. Eng. & Biotech. News 2010,14-15.). It was found that by changing the pyrrolopyrazine to apyrrolopyrimidine, a significantly improved in vitro safety profile wasachieved. Comparing the matched pair Comparator B (pyrrolopyrazine) withComparator D (pyrrolopyrimidine), a significant improvement in both theCFU erythroid and myeloid progenitor cells is observed. This also holdsfor the aminopyridine urea matched pairs possessing a thiophenefootpocket, Comparator C (pyrrolopyrazine) and Comparator E(pyrrolopyrimidine). Although the pyrimidine brought with it an improvedsafety profile, both unsubstituted pyrimidine compounds Comparator D andComparator E showed significant inhibition of CYP2D6 at 10 μM. However,substituting on the pyrimidine ring at the position between the nitrogenatoms, Compound 2 (methyl-substituted pyrrolopyrimidine) showed nosignificant inhibition of either CYP2D6 or CYP3A4 at 10 μM.

These results evidence that slight chemical modifications such as thewalking of a nitrogen atom one position on a ring in Comparators B and Dand replacing hydrogen for methyl (Comparators E and Compound 2) producedramatic increases in safety.

TABLE 2 Comparison of in vitro profile of Compound 2 to multiplecomparators. HDAC2 HDAC SY5Y cell recombinant % CYP lysate enzymaticIC50 inhibition CFU % Control assay (μM) @ 10 μM: remaining @ 10 μMComp. Structure IC50 (μM) HDAC2 HDAC1 2D6 3A4 Erythroid Myeloid A

0.369 0.142 0.027 34 −22 0 0 B

0.485 0.475 0.119 9 1.5 9 25 C

0.279 0.301 0.095 0.2 1 20 20 D

0.331 0.627 0.239 85 2 47 84 E

0.278 0.511 0.142 50 −17 54 83 2

0.577 0.434 0.133 −5 1 27 59

In addition to the substitution between pyrimidine nitrogen atomsimproving the CYP inhibition profile of pyrrolopyrimidine compounds, thePK profile of Compound 2 is improved relative to unsubstitutedComparator E as well. See Table 3. The methyl-pyrimidine of Compound 2provides distinct PK benefits over the unsubstituted pyrimidine analog,displaying a longer half-life, lower clearance, higher bioavailability,and a >5-fold higher brain exposure. These results evidence that slightchemical modifications also produce substantial benefits in PK.

TABLE 3 Comparison in PK profiles of unsubstituted pyrimidine andmethyl-substituted pyrimidine matched pairs. Comparator E Compound 2Structure

Mouse IV (1 mpk): T1/2 (hr) 0.152 0.839 Mouse IV (1 mpk): Cl (L/hr/kg)8.33 4.18 Mouse IV-PO (1/10 mpk): F (%) 49 100 Mouse PO (1/10 mpk): T1/2(hr) 0.557 1.05 Mouse PO (10 mpk): Brain 100 583 Cmax (ng/g) (free Cmax,ng/g) Mouse PO (10 mpk): Free brain 35 173 Cmax (ng/g)

Similar advantages were gained by exploring the effects of regioisomersand substitution patterns on pyrrolopyridine ureas of diaminopyridines.For example, while pyrrolopyridine compound Comparator F showed good invitro potency, it showed high levels of CYP2D6 inhibition and extremelylow solubility. See Table 4. Moving the pyridine nitrogen of thepyrrolopyridine (Comparator G) was found to improve solubility. However,both CYP2D6 and CYP3A4 were inhibited at high levels. It was found thatadding a methyl substituent adjacent to the pyridine nitrogen of thepyrrolopyridine improved the CYP inhibition profile toward 2D6 and 3A4somewhat, while maintaining the potency and solubility (Comparator H).The electron withdrawing methoxymethyl substitution adjacent to thepyridine nitrogen (Comparator H) improved the CYP 2D6 and 3A4 inhibitionprofile even further, again maintaining the desirable potency andsolubility profile. Similar results were found between the mono-fluoroand di-fluoro analogues of Comparator I and Compound 1, with Compound 1displaying slightly reduced solubility. See Table 4.

However, Compound 1 vastly out performed Comparator I with respect to invitro safety, despite the only one halogen atom difference. See Table 5.A significant benefit was realized in both the erythroid and myeloidprogenitor cell lineages upon treatment with the 2,4-difluorosubstituted Compound 1 relative to Comparator I.

TABLE 4 Comparison of in vitro profile of Compound 1 to multiplecomparators. HDAC recombinant % CYP HDAC2 SY5Y enzymatic IC50 inhibition@ cell lysate assay (μM) 10 μM: Solubility Cmp. Structure IC50 (μM)HDAC2 HDAC1 2D6 3A4 (μM) F

0.394 0.304 0.079 62 −4 1 G

0.639 0.335 0.143 94 65 55 H

0.621 0.214 0.090 57 28 78 I

0.666 0.276 0.122 2 33 121 1

0.777 0.510 0.326 −1 27 27

TABLE 5 In vitro CFU assay data for Compound 10 and Comparator IComparator I Compound 1 Structure

CFU % Control remaining 23 57 @ 10 μM: Erythroid CFU % Control remaining59 86 @ 10 μM: Myeloid

Table 6 below shows the results from the brain and plasma exposurefollowing intravenous (IV) and Oral (PO) administration of compounds inmice.

TABLE 6 Projected Projected Brain Cmax free brain @ 10 mpk @ 10 mpk ng/guM (*scaled (*scaled for for IV PK IV PK Cl Structure comparison)comparison) T ½ (hr) (L/hr/kg) Comparator D

 158* 0.111* Not available Not available 1

2460 1.04 2.13 PO = 1.97 0.681

Effects of 14-days of oral treatment with low doses of compounds ondendritic spine morphology in dorsal hippocampus (CA1) of Wild Type Mice

Compounds were evaluated to determine whether sub-chronic treatmentcould produce changes in dendritic spine morphology in the CA1 region ofthe dorsal hippocampus in wild type (WT) mice. Compounds wereadministered orally to wild type mice, daily for 14 days. Doses werechosen based on pharmacokinetic data, brain exposure, and potency.Effects of compounds treatments on dendritic spine morphology in the CA1region of the dorsal hippocampus were then evaluated.

Methods: In-life

Male C57BL/6J mice (7-8-weeks old, n=7 per group) were dosed orally withRodin compounds or vehicle (20% HPRCD) daily for 14 days. Doses forcompounds were chosen based on exposure data from pharmacokineticexperiments. Doses of some compounds were chosen to determinenon-efficacious doses, as an extension of earlier studies showingincreases in dendritic spine density after treatment with Compound 2 (atdoses of 1, 3, 6 and 20 mg/kg/day), Compound 1 (10 mg/kg/day). Mice weresacrificed 24 hours after the last dose, and underwent transcardialperfusion for preparation of brain samples.

Perfusion and Brain Sampling Method

Mice were anesthetized with chloral hydrate (4% chloral hydrate insaline, 10 ml/kg) before undergoing transcardial perfusion with 4% PFAin 1×PBS (pH7.4, room temperature) at 20 mL/min for 54 secondsImmediately after the perfusion, mice were decapitated and their brainextracted. Brains were postfixed in scintillation vials containing 4%PFA (5-10 ml) for 4 min. Brains were sectioned using a tissue vibratome(Leica VT1000) to collect sections (300 μm thick) from the anterior toposterior extremes of each brain.

Ballistic Dye Labeling and Microscopy

Superresolution laser-scanning confocal microscopy (Zeiss LSM880,Airyscan) was performed using a 63× objective (1.42 NA) to scanindividually labeled neurons at high resolution (scan resolution =0.06um pm/pixel; axial resolution =0.06 um μm/focal step). Target neuronswere identified in the brain region of interest by epifluorescencenavigation using anatomical location and cell morphology. Microscopy wasperformed blind to experimental conditions. A minimum of 7 mice weretested in each experimental condition. A minimum of 5 samples per mouse(range=5-6) were measured for each segment.

ESP Dendritic Spine Analysis and Assessment of Dendritic MembraneIntegrity

Blind deconvolution (AutoQuant) was applied to raw three-dimensionaldigital images which were then analyzed for spine density and morphologyby trained analysts. Individual spines were measured manually for (a)head diameter, (b) length, and (c) neck thickness from image Z-stacksusing custom-built Afraxis ESP software. Each dendrite was analyzed by 3(on average) independent analysts. Automated image assignment softwaredistributed images to analysts in a randomized manner and ensured thateach analyst performed measurements of near equal numbers of dendritesper group. Analysts were blinded to all experimental conditions(including treatment, brain region, and cell type). Statistical analysisof interanalyst variability for each dendrite was examined on-line andused to eliminate dendrites that did not meet interanalyst reliabilitycriteria: For spine density and spine morphological classification, dataacross analysts were averaged to report data for each dendrite.

Dendritic Sampling Positions

-   dHIPP, CA1 Apical 2° (0-50 μm):-   Brain region: Dorsal Hippocampus (dHIPP)-   Cell type: CA1 pyramidal neuron (CA1)-   Branch type: Apical-   Branch order: Secondary (2°)-   Sample position: 0-50 μm from branchpoint

Each identified dendritic spine was measured for (a) spine length, (b)spine head diameter, and (c) neck width. Population distributions ofeach measure were compiled for each dendritic sample and pooled bygroup. Raw dendritic spine morphometric values (spine length, headdiameter, neck width) were assembled into a scheme used to describeclassic spine phenotypes (e.g. mushroom, stubby, etc.). Total spinedensity was also reported as the sum of the density of all subclasses.

Results

General Observations. The tissue processing demonstrated no observablepathological indications, including abnormal disruption of somaticmembranes, dendritic blebbing, or abnormal modifications of dendritediameters for the target cell type or other cell types within the brainregions tested. For adequate study to study comparisons, total spinedensity data were normalized to % vehicle levels

${\left. 1 \right)\mspace{14mu} \% \mspace{14mu} {Vehicle}} = {\frac{\left( {{Spine}\mspace{14mu} {density}\mspace{14mu} {or}\mspace{14mu} {SV}\; 2A\mspace{14mu} {puncta}} \right)}{\left( {{Average}\mspace{14mu} {Vehicle}\mspace{14mu} {spine}\mspace{14mu} {density}\mspace{14mu} {or}\mspace{14mu} {SV}\; 2A\mspace{14mu} {puncta}} \right.} \times 100}$

Data were then reported as mean+/−SEM of total spine density (% increasefrom Vehicle). The results are reported in Table 7.

TABLE 7 Effects on spine morphology in WT mice after 14 days of dosing.Total Spines Thin Spines Dose (% increase (% increase Compound Structure(mg/kg) over vehicle) over vehicle) 1

0.1 0.3 1   3   10   12.2  21.3* 34.6* 26.7* 25.1* 13.8  23.2* 45.1*25.3* 19.9* 2

0.1 1   3   6   20   8.1 21.4* 24.7* 15*    −0.12   11.3  21.2  32.2*35.4* 37.4* *significantly different from vehicle with p value <0.05using a one-way ANOVA followed by a Dunnett's postHoc analysis

The contents of all references (including literature references, issuedpatents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated herein in their entireties by reference. Unless otherwisedefined, all technical and scientific terms used herein are accorded themeaning commonly known to one with ordinary skill in the art.

1. A compound of the formula:

or a pharmaceutically acceptable salt thereof.
 2. A pharmaceuticalcomposition comprising the compound of claim 1, or a pharmaceuticallyacceptable salt thereof; and a pharmaceutically acceptable carrier. 3.(canceled)
 4. A method of treating a condition in a subject selectedfrom a neurological disorder, memory or cognitive function disorder orimpairment, disorder of learning extinction, fungal disease orinfection, inflammatory disease, hematological disease, psychiatricdisorders, and neoplastic disease, comprising administering to thesubject in need thereof an effective amount the compound of claim
 1. 5.The method of claim 4, wherein the condition is: a. a cognitive functiondisorder or impairment associated with Alzheimer's disease, Huntington'sdisease, seizure induced memory loss, schizophrenia, Rubinstein Taybisyndrome, Rett Syndrome, depression, Fragile X, Lewy body dementia,vascular dementia, fronto-temporal lobar degeneration (FTLD), ADHD,dyslexia, bipolar disorder and social, cognitive and learning disordersassociated with autism, traumatic head injury, traumatic brain injury(TBI), multiple sclerosis, attention deficit disorder, anxiety disorder,conditioned fear response, panic disorder, obsessive compulsivedisorder, posttraumatic stress disorder (PTSD), phobia, social anxietydisorder, substance dependence recovery, Age Associated MemoryImpairment (AAMI), Age Related Cognitive Decline (ARCD), ataxia,Parkinson's disease, or Parkinson's disease dementia; or b. ahematological disease selected from acute myeloid leukemia, acutepromyelocytic leukemia, acute lymphoblastic leukemia, chronicmyelogenous leukemia, myelodysplastic syndromes, and sickle cell anemia;or c. a neoplastic disease; or d. a disorder of learning extinctionselected from fear extinction and post-traumatic stress disorder.
 6. Themethod of claim 5, wherein the condition is Alzheimer's disease,Huntington's disease, fronto-temporal lobar degeneration (FTLD)Friedreich's ataxia, post-traumatic stress disorder (PTSD), Parkinson'sdisease, or substance dependence recovery.
 7. A method of treating acondition in a subject selected from a neurological disorder, memory orcognitive function disorder or impairment, neurological disorder withsynaptic pathology, disorder of learning extinction, fungal disease orinfection, inflammatory disease, hematological disease, psychiatricdisorders, and neoplastic disease, comprising administering to thesubject in need thereof an effective amount a compound of the Formula:

or a pharmaceutically acceptable salt thereof.
 8. The method of claim 7,wherein the condition is: a. a cognitive function disorder or impairmentassociated with Alzheimer's disease, Huntington's disease, seizureinduced memory loss, schizophrenia, Rubinstein Taybi syndrome, RettSyndrome, depression, Fragile X, Lewy body dementia, vascular dementia,fronto-temporal lobar degeneration (FTLD), ADHD, dyslexia, bipolardisorder and social, cognitive and learning disorders associated withautism, traumatic head injury, traumatic brain injury (TBI), multiplesclerosis, attention deficit disorder, anxiety disorder, conditionedfear response, panic disorder, obsessive compulsive disorder,posttraumatic stress disorder (PTSD), phobia, social anxiety disorder,substance dependence recovery, Age Associated Memory Impairment (AAMI),Age Related Cognitive Decline (ARCD), ataxia, Parkinson's disease, orParkinson's disease dementia; or b. a hematological disease selectedfrom acute myeloid leukemia, acute promyelocytic leukemia, acutelymphoblastic leukemia, chronic myelogenous leukemia, myelodysplasticsyndromes, and sickle cell anemia; or c. a neoplastic disease; or d. adisorder of learning extinction selected from fear extinction andpost-traumatic stress disorder.
 9. The method of claim 8, wherein thecondition is Alzheimer's disease, Huntington's disease, fronto-temporallobar degeneration (FTLD), Friedreich's ataxia, post-traumatic stressdisorder (PTSD), Parkinson's disease, or substance dependence recovery.